Mutually facilitated co-transport of two different viruses through reactive porous media

Water Research 123 (2017) 40e48

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Mutually facilitated co-transport of two different viruses through reactive porous media Shuang Xu a, e, Ramesh Attinti b, Elizabeth Adams c, Jie Wei d, Kalmia Kniel d, Jie Zhuang a, f, *, Yan Jin b, ** a Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, Liaoning, 110016, China b Department of Plant and Soil Sciences, University of Delaware, Newark, DE, 19716, USA c Delaware Biotechnology Institute, University of Delaware, Newark, DE, 19716, USA d Department of Animal and Food Sciences, University of Delaware, Newark, DE, 19716, USA e University of Chinese Academy of Sciences, Beijing, 100039, China f Department of Biosystems Engineering and Soil Science, University of Tennessee, Knoxville, TN, 37996, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 February 2017 Received in revised form 11 June 2017 Accepted 14 June 2017 Available online 18 June 2017

In this study, we quantified transport and retention behaviors of a human pathogenic virus (Adenovirus 41, abbreviated as Ad-41) and a model bacteriophage (fX174) through metal oxide-removed and goethite-coated sand under saturated flow conditions. Two sets of bench scale column experiments were conducted: transport of each type of viruses alone and co-transport of both viruses. All experiments were conducted at pH 7.5 using 2 mM artificial ground water (AGW) buffer as background solution. Experimental results revealed that goethite-coated sand (i.e., reactive sand) retained much more viruses relative to metal oxide-removed sand (i.e., non-reactive sand), with the difference more pronounced for fX174 (effluent concentration decreased by 92.1%) than for Ad-41 (effluent concentration decreased by 59.7%). Interestingly, lower retention of both viruses on goethite-coated sand was observed when they co-existed in the influent. The mutual promotion to the transport of the two viruses may be attributed to attachment site competition and steric hindrance effect (illustrated in the graphical abstract). Mass recovery results revealed that fX174 was largely reversibly attached, whereas Ad-41 was mostly irreversibly bound or inactivated. Force measurements using atomic force microscopy (AFM) demonstrated that fX174 had doubled affinity to goethite-coated sand compared with Ad-41, consistent with the transport behavior of each type of viruses when they existed alone. As expected, the extended DLVO theory cannot accurately estimate interfacial energy profiles for viruses and reactive sand that have heterogeneous surface properties (i.e., roughness and charge heterogeneity). The results of this study clearly demonstrate that caution must be taken when applying laboratory results, which are generally obtained from experiments employing a single virus species, to predict the mobility and environmental risks in natural systems where multiple agents are present. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Adenovirus Steric interaction XDLVO Co-transport Goethite Adhesion force

1. Introduction Water contamination by pathogenic viruses is a serious public health concern and may bring about economic and societal burdens

* Corresponding author. Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, Liaoning, 110016, China. ** Corresponding author. E-mail addresses: [email protected] (J. Zhuang), [email protected] (Y. Jin). http://dx.doi.org/10.1016/j.watres.2017.06.039 0043-1354/© 2017 Elsevier Ltd. All rights reserved.

(Ogorzaly et al., 2015; Wyn-Jones et al., 2011; Borchardt et al., 2007). A complete understanding of virus behavior (e.g., retention, survival, and transport) is therefore prerequisite for accurate assessment of the risks associated with groundwater contamination by viruses, as well as the design of effective prevention and remediation strategies (Chattopadhyay and Puls, 2000). The transport and infectivity of viruses are mainly affected by their attachment to solid surfaces, and mechanisms responsible for the attachment involving electrostatic interactions (Wong et al., 2014; Zhuang and Jin, 2008), van der Waals interactions (Fuhs et al., 1985; Moore et al., 1982), steric interactions (Pham et al., 2009;

S. Xu et al. / Water Research 123 (2017) 40e48

41

Yuan et al., 2008), and hydrophobic effect (Armanious et al., 2016; Bales et al., 1991). Electrostatic interactions play a primary role for attachment to charged surfaces and are repulsive/attractive if the viruses and surfaces carry same/opposite surface charges, respectively (Fuhs et al., 1985). Virus attachment onto solid surfaces in laboratory experiments is mostly investigated from solutions that contain only a single virus species (Kokkinos et al., 2015; Park et al., 2014; Shi et al., 2012; Syngouna and Chrysikopoulos, 2011; Attinti et al., 2010; Corapcioglu et al., 2006; Schijven et al., 2002). While allowing for selectively studying virus-surface interactions, this experimental approach does not account for potential interactions between more than one virus species and how such interactions in turn affect their retention and transport behaviors in natural subsurface environments where multiple viruses are likely to coexist. A large number of waterborne viruses are electronegative at circumneutral pH (Michen and Graule, 2010) and are expected to attach to positively charged surfaces (i.e. iron oxides). It is thus reasonable to expect competitive attachment to solid surfaces between viruses. A series of scenarios are plausible for the mutual transport effect of viruses. Firstly, negatively charged viruses directly compete for positively charged surfaces (favorable sites) due to electrostatic attraction/adhesion force. This scenario predicts competitive suppression of virus attachment and, as a consequence, increasing virus mobility during their co-transport. Secondly, once viruses are attached to the sites, they can block the attachment sites and even adjacent sites through steric interaction, which in turn hinder the attachment of incoming viruses. Thirdly, the attachment of electronegative viruses on favorable sites can increase the overall electronegativity of solid surfaces, and thus the increased electrostatic repulsion between the attached and incoming viruses leads to increased transport. In addition, if one species of electronegative viral particles carry positively charged sites (e.g. fibers with high isoelectric point), other negatively charged viruses may attach to these favorable sites to co-deposit on sand surfaces. While most studies on virus transport have focused on the behavior of a single species, a fundamental understanding of the mutual interactions of different viruses is, however, required to accurately predict the fate and transport of viruses in natural subsurface environment. The main objective of this study was to provide a systematic analysis of the co-transport behavior of viruses (human pathogenic virus Ad-41 and a model bacteriophage fX174) through reactive porous media. The transport and retention of each virus alone were quantified to compare with their co-transport behavior. Adhesion forces between the viruses and goethite-coated sand were measured using atomic force microscopy (AFM). Effectiveness of the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory for estimating virus-sand interfacial behaviors was addressed.

1000 g oxide
removed sand and 200-mL NaHCO3 were added. During the mixing process, the solution was stirred vigorously and aerated. Oxidation was complete when the color of the suspension changed from greenish
blue to ochre. The pH during the oxidation was controlled at 7.0 using NaHCO3 buffer. After the sand had been coated by goethite, it was rinsed with copious amount of deionized distilled water (DDW) to remove any electrolyte residues in the solution and on sand surfaces. The goethite
coated sand was then air
dried and stored in clean plastic bottles at room temperature (21 ± 1  C) until use. Scanning electron microscopy (SEM, S4700, Hitachi High Technologies America, Inc.) and energy-dispersive Xray spectroscopy (EDS) analyses were performed to examine morphology and to confirm the presence of Fe coating on the sand. Zeta potentials of the coated sand were measured using a Zetasizer (ZEN 3600, Malvern Instruments Ltd.) in an artificial ground water (AGW) solution, which is composed of 2 mM CaCl2, MgCl2$6H20, KCl and NaHCO3 with pH 7.5, the same as used for transport experiments following the procedure described previously (Attinti et al., 2010). The measured zeta potentials and referenced water contact angles of viruses, oxide-removed sand, and goethite-coated sand are provided in Table 1.

2. Materials and methods

Transport experiments were conducted under saturated flow conditions using polycarbonate columns (10 cm in length and 3.5 cm in inner diameter), a setup similar to that illustrated in Jin et al. (1997). Each column was wet-packed by pouring the experimental sand into de-aired AGW solution-filled column at 0.5 cm increments while stirring with an autoclaved glass rod. The bulk density of the packed sands was 1.78 g/cm3, and the porosity was 0.33 (based on a particle density of 2.65 g/cm3). Each column was flushed upward with 10 pore volumes (PVs) of autoclaved de-aired AGW buffer solution to remove air bubbles and to establish a steady-state flow at a defined flow velocity of 0.103 cm/min. To observe the flow conditions in the packed column, 4 PVs of nonreactive tracer bromide solution (25 mg/L) was introduced and then the column was flushed with another 4 PVs of AGW buffer solution. Finally, the input was switched to AGW suspension that

2.1. Porous media Accusand (Unimin, Le Sueur, MN), a mixture of several grain sizes (9% of 0.1e0.25 mm, 69.8% of 0.25e0.5 mm, and 21.2% of 0.5e1.0 mm), was used for the column transport experiments. The sand was treated to remove impurities and metal oxides using citrate buffer solution following the procedure described in Chu et al. (2001). The treated sand is referred to as oxide-removed sand (i.e., non-reactive sand). Preparation of goethite-coated sand was carried out according to the method described by Cheng et al. (2004), and a brief description is provided here. Briefly, 20 g of unoxidized crystals of FeCl2$4H2O was dissolved in 1-L distilled water through which N2 had been bubbled through for 30 min. Then,

2.2. Viruses Two viruses, human pathogenic Adenovirus 41 (Ad-41) and a model bacteriophage (fX174) were employed in this study. Ad-41 is a double-stranded, icosahedron DNA virus with a particle size of 70e90 nm and contains 240 hexons, 12 pentons, and 12 fibers that extend from each penton base (Favier et al., 2002, 2004). The number of short fibers was 6 times more than that of long fibers (Song et al., 2012) and the isoelectric point was 9.13 and 7.51 for short and long fibers, respectively (Favier et al., 2002; Yeh et al., 1994). Adenovirus concentrations have been reported to range from 1.5  102 to 2  105 GC/mL in freshwater (Hassard et al., 2016; Staggemeier et al., 2015). fX174 is a spherical single-standard DNA virus with an effective diameter of 23 nm and an isoelectric point of 6.6 (Ackerman and Dubow, 1987; Syngouna and Chrysikopoulos, 2013). Both viruses were centrifuged at 61,500g for 4 h at 4  C using an ultra-centrifuge (Beckman, SW28 rotor) to obtain concentrated virus titers. fX174 and Ad-41 were enumerated using the plaque forming unit (PFU) assay method (Adams, 1959) and tissue culture infective dose (TCID50) method (Enriquez et al., 1995), respectively. The detection limits of fX174 and Ad-41 were set to be 10 PFUs per plate and 101.2 TCID50/mL, respectively. Preliminary batch inactivation experiments under the experimental conditions showed no significant virus inactivation for the duration of the experiments. 2.3. Transport experiments

42

S. Xu et al. / Water Research 123 (2017) 40e48

Table 1 Constants and inputs for XDLVO and steric force calculation. Constant/Input

Symbol

Value

Unit

Reference

Fluid approach velocity Porosity of bed Radius of Ad-41 Radius of fX174 Radius of sand Length of the column Water contact angle of Ad-41 Water contact angle of fX174 Water contact angle of oxide-removed sand Water contact angle of goethite-coated sand Zeta potential of Ad-41 Zeta potential of fX174 Zeta potential of oxide-removed sand Zeta potential of goethite-coated sand Avagodras number Molar volume of water

U ε av1, r1 av2, r2 as L

6.2E-02 33.0 3.5E-08 1.2E-08 0.2E-08 0.1 33.0 ± 0.5 26.0 ± 1.7 16.0 ± 1.1 62.0 ± 2.1
23.6 ± 1.2
31.3 ± 1.2
39.5 ± 0.8 4.1 ± 0.6 60.2Eþ22 1.8E-05

m/h % m m m m degree degree degree degree mV mV mV mV mol
1 m3/mole

This study This study This study This study This study This study Wong et al. (2012) Attinti et al. (2010) Attinti et al. (2010) Attinti et al. (2010) This study This study This study This study e e

4.9E-01 3.1E-08 8.3E-08 1350.0 64.0

e m kg/m2 kg/m3 kg/mole

Penrod et al. (1996) Ruigrok et al. (1990) This study Quillin and Matthews (2000) Wong et al. (2012)

Flory-Huggins solvency parameter Length of the fiber Maximum surface concentration Density of fiber Molecular weight of fiber

qAd-41 qfX174 qoxide-removed sand qgoethite-coated sand

εAd-41 εfX174 εoxide-removed sand εgoethite-coated sand NA V

c d Gmax

rP

Mw

contained fX174 (106 PFU/mL) or Ad-41 (102e104 TCID50/mL) or both viruses, followed by injection of several PVs of virus-free AGW buffer solution. The input suspension was introduced from the bottom of the column through a peristaltic pump and the effluent samples were collected from the top exit of the column using a fraction collector. Ion chromatography (IC, Dionex Corporation, Sunnyvale, CA) was used to determine bromide concentration in the effluent samples. Two sets of column transport experiments were performed, with Ad-41 or fX174 alone or co-existing in the influent. All the experiments used AGW (pH 7.5 in 2 mM) as background buffer solution and were run in a large refrigerator at ~4  C to minimize virus inactivation. To examine whether the viruses retained in the columns are reversible and to estimate the total mass recovery of the viruses, beef extract solution (weight/ volume 3%, pH 9.5), a mixture of high ionic strength enzymatic acids made from hydrolyzed beef, was used to elute the column immediately after virus input was ceased (Gerba, 1984; Jin et al., 1997; Zhuang and Jin, 2003). Total mass recovery was defined as the ratio of the total number of particles in the effluent during both breakthrough and elution phase divided by total number of virus particle input.

using the method described by Hutter and Bechhoefer (1993), similar to those provided by the manufacturer, were used to determine the force (F) of the interactions using the Hooke's law (Gaboriaud and Dufrene, 2007). Each average value of measured virus
sand interaction force represents measurements taken at four different locations with 250 measurements within each location. 2.5. Surface coverage rate estimation For irreversible virus attachment occurring in a packed column of spherical collectors, coupled with nonexistence of multilayer attachment of virus due to ripening, the fractional surface coverage q (i.e., number of cells m
2  pa2v ) of the collector, defined as the area of deposited cells divided by the porous media surface area in the column, can be estimated as a function of pore volumes by integrating the breakthrough curve (Liu et al., 1995; Rijnaarts et al., 1996):

q¼

pa2v Uas C0

2.4. AFM force measurements A Bioscope II atomic force microscopy (AFM, Bruker Instruments Inc.) mounted on an Axiovert 200 inverted fluorescent microscope (Carl Zeiss Inc.) operating in the contact mode was used to measure virus
sand force interactions. The silicon nitride AFM cantilevers (Bruker Probes Inc.) were first cleaned using a piranha solution (70:30 sulphuric acid: hydrogen peroxide) and then silanized using APTES (1.9 mL EtOH, 0.1 mL water and 80 mL APTES for 1 h at 21  C). Any unbound silane was removed by washing the cantilevers inEtOH. The cantilevers were then dried at 100  C for 5 min. Virus particles were attached to the cantilevers using 2.5% glutaraldehyde solution (AFM cantilevers were incubated in 50 mL of virus suspension and 50 mL of 2.5% glutaraldehyde solution at room temperature for 1 h). The cantilevers were then rinsed with the AGW buffer solution. Goethite
coated sand was fixed to microscopic glass slides with epoxy resin and allowed to cure overnight at room temperature. The virus coated cantilevers were then used to quantify the adhesion force between virus and goethite-coated sand. The measured spring constants of virus coated cantilevers

Z t 0

1


C C0

 dt

3Lð1
εÞ

a ¼ a0 ð1
BqÞ a0 ¼


4 ac lnðC=C0 Þ 3 Lnð1
εÞ

(1) (2) (3)

where av (m) and as (m) are virus and collector radius, respectively, U (m/s) is the fluid approach velocity, L (m) is the length of the column, ε (
) is the porosity of packed sand, a (
)is the collision efficiency, a0 (
) is the clean sand collision efficiency, B (
) is the blocking factor, q (
) is the fraction of surface area covered, and C0 (N m
3) and C (N m
3) are the virus input and effluent particle number concentrations, respectively. 2.6. Extended DLVO and steric interactions According to the extended DLVO (XDLVO) theory, the surface potential energy (FXDLVO) is calculated by the sum of potential energy contributed by van der Waals(FVdw), electrostatic double layer

S. Xu et al. / Water Research 123 (2017) 40e48

(FEdl), Lewis acid-base (FAB), and Born interactions (FBorn). Details of the calculations of FVdw, FEdl, FBorn, and FAB can be found in a previous study (Xu et al., 2016). The Hamaker constant values for oxide-removed sand-virus-water, goethite-coated sand-virus-water are 3.4  10
21 J (Bradford and Torkzaban, 2008) and 6.0  10
21 J (Foppen et al., 2006), respectively. In addition, steric interaction was reported to have a significant influence on the attachment behavior of human adenovirus (Wong et al., 2012), thus the steric energy was added to the XDLVO energies to calculate the total interaction energy between Ad-41 and sand surfaces:

Fsteric ¼ Fosm þ Felas

(4)

Fosm ¼

  2p r F2P NA 1
c ðd
hÞ2 2 V

(5)

FP ¼ 3

G r2 h max i rP ðd þ rÞ3
r3

(6)

Felas

"        # 2pr NA FP d2 rP 2 1 h 3 h h h
þ ln ¼
3 6 d 2d d d MW (7)

where Fosm (KBT) is osmotic interaction energy, FP (
) is the volume fraction of the fiber, Felas (KBT) is elastic interaction energy, V (m3/mol) is the molar volume of water, c (
) is Flory-Huggins solvency parameter, d (m) is the length of the fiber, MW (kg/mol) is the molecular weight of the fiber, rP (kg/m3) is the density of the fiber, and Gmax (kg/m2) is the maximum surface concentration. The values of relevant parameters are provided in Table 1. 3. Results and discussion 3.1. Virus transport mediated by surface properties Surface properties of porous media have a key influence on virus transport. The retention of fX174 and Ad-41 was minimal during their transport through oxide-removed sand (Fig. 1a). Virus breakthrough occurred at ~1 PV, with average maximum relative concentrations (max C/C0) ~0.93 for fX174 and ~0.83 for Ad-41 and effluent percentages ~93% for fX174 and ~80% for Ad-41, respectively (Table 2). The results indicate unfavorable interactions due to electrostatic repulsions between the like-charged viruses and sand

43

particles. Compared to the oxide-removed sand, goethite-coated sand exhibited much greater retention capacity for both viruses (Fig. 1b), with max C/C0 ~0.03 for fX174 and ~0.3 for Ad-41, respectively, and effluent percentages decreasing to ~1% for fX174 and ~20% for Ad-41 (Table 2). These large differences can be attributed to changes of sand surface properties after coating with goethite, such as surface roughness and charges (Zhuang and Jin, 2008). Shen et al. (2011) found that the sharp asperities on sand surfaces increased colloid retention in primary minima by reducing the energy barrier, and the existence of large valleys facilitated colloid deposition in secondary minima by increasing the energy barriers as well as the secondary-minimum depth. In this study, goethite coating increased the water contact angle of sand from 16.0 ± 0.1 to 62.0 ± 2.1 (Table 1), which is likely caused by the increase in surface roughness of the sand (Zhao et al., 2014). In addition, goethite coating increased the surface zeta potential of the oxide-removed sand from
39.5±0.8 mV to 4.1±0.6 mV, thus changing virus-sand interactions from repulsion to attraction. Mass recovery results indicated that goethite-coated sand removed more of both viruses than oxide-removed sand. The mass recovery results showed that ~98% of injected fX174 and ~88% of injected Ad41 were recovered during the transport and elution processes from oxide-removed sand, while only ~72% of fX174 and ~20% of Ad-41 recovered from goethite-coated sand. The protonated, positivelycharged surface sites on goethite can cause irreversible virus attachment/inactivation, nearly all retained Ad-41 were inactive on goethite-coated sand, while 60% of Ad-41 remained inactive on oxide-removed sand. This discrepancy was not obvious for fX174, only ~29% of retained fX174 became inactive both on oxideremoved sand and goethite-coated sand. Straining and wedging were not considered important mechanisms for virus retention in this study, as the ratio of virus to collector diameter is far below the suggested threshold of 0.003 (Bradford and Bettahar, 2006). Surface properties of viruses also determine their transport. The different retention and transport behaviors of fX174 and Ad-41 through either oxide-removed sand or goethite-coated sand are likely due to the variation of their properties. fX174 (zeta potential
31.3 ± 1.2 mV) has higher electro-negativity than Ad-41 (zeta potential
23.1 ± 1.2 mV) in the experimental AGW solution (pH 7.5 in 2 mM). Thus, fX174 experienced greater electrostatic repulsion from oxide-removed sand (reducing retention) and greater electrostatic attraction from goethite-coated sand (enhancing retention) than Ad-41. In addition, the fibers of Ad-41 were reported to control the affinity and how Ad-41 interacts with surfaces (Shi et al., 2012). The high isoelectric points on Ad-41 fibers (9.13 for short fibers and 7.51 for long fibers) indicated that

Fig. 1. Breakthrough of fX174 and Ad-41 from non-reactive and reactive sand media.

44

S. Xu et al. / Water Research 123 (2017) 40e48

Table 2 Estimated mass recovery of fX174 and Ad-41. Parameters

Individual transport Oxide-removed sand

fX174 Effluent (%) Removal (%) Elution (%)c Inactivation (%)d Recovery (%)e Parameters

Effluent (%) Removal(%) Elution (%) Inactivation (%) Recovery (%) a b c d e

a

Goethite-coated sand Ad-41 (1.1  10 )

fX174

Ad-41 (1.1  104)

fX174 (long term)

79.8 20.2 8.0 60.4 87.8

0.6 99.4 71.1 28.5 71.7

20.1 79.9 0.3 99.6 20.4

72.5 27.5 8.4 69.5 80.9

b

92.7 7.3 5.2 28.8 97.9

4

Co-transport in goethite-coated sand

fX174

Ad-41 (8.6  102)

fX174

Ad-41 (1.1  104)

fX174

Ad-41 (4.8  104)

56.3 43.7 23.8 45.5 80.1

0.7 99.3 6.4 93.6 7.1

64.8 35.2 20.2 42.6 85.0

29.3 70.7 1.5 97.9 30.8

70.6 29.4 15.1 18.6 85.7

34.0 66.0 0.1 99.8 34.1

1  106 PFU/mL of fX174 was used in all experiments. The concentration unit of Ad-41 in the input was TCID50/mL in all experiments. Refers to the breakthrough percentage of viruses during the elution of beef extract. Inactivation % ¼ (Removal % - Elution %)/Removal %. Recovery % ¼ Effluent % þ Elution %.

the fibers were positively charged under the experimental conditions (pH 7.5) (Favier et al., 2002; Yeh et al., 1994), thus the electropositive fibers likely contributed to the greater retention of Ad41 on the opposite-charged oxide-removed sand and a lower retention on the like-charged goethite-coated sand. Conspicuously, the smaller-sized, moderately hydrophilic fX174 exhibited a higher mass recovery (Table 2), likely due to insignificant size exclusion effect (Ghanem et al., 2016) and decrease in hydrophobicityinduced Lewis acid-base attraction between fX174 and sand surfaces (Xu et al., 2016). In addition, the much higher percentage of irreversible attachment/inactivation of Ad-41 (~100%) compared to fX174 (~29%) suggested that fX174 was retained mainly via reversible attachment, whereas Ad-41 was mostly irreversibly attached or inactivated on goethite-coated sand. This difference might be because the hair fibers of Ad-41 are more susceptible to damage than the surface protein of fX174. However, this speculation needs further study for confirmation.

Fig. 2. Adhesion force between virus and goethite-coated sand measured at different separation distances. The negative force values mean attractive interactions between virus and sand.

The above macroscopic transport behaviors were verified by microscopic measurements of adhesion forces using AFM. Fig. 2 shows the interaction forces measured between viruses (fX174 and Ad-41coated on AFM silicon nitride tips) and goethite-coated sand. fX174-sand surface interactions were detected, beginning at ~130 nm and reached the highest attractive adhesive force at 6.8 nN, while for the Ad-41 a lower attractive adhesive force of 4.5 nN was detected, beginning at ~70 nm from the surface. The average adhesion force at multiple separation distances on the goethite-coated sand was 5.6 ± 0.6 nN for fX174 and 3.0 ± 0.4 nN for Ad-41. These results indicate a greater adhesion force of fX174 on goethite-coated sand than Ad-41, consistent with the observation from the column experiments where the effluent percentage of fX174 (1%) was lower than Ad-41 (20%) through the goethitecoated sand. The above transport behaviors of the two viruses were further evaluated according to the energy profiles estimated using the XDLVO theory and steric effect (Fig. 3). The nonexistence of primary energy barriers, primary minima, and secondary energy minima suggest minimal attachment of both viruses on the oxide-removed sand, consistent with the breakthrough results of viruses (Fig. 1a). In contrast, there was no primary energy barriers and secondary energy minimum on the goethite-coated sand, suggesting that attractive forces dominated both phages. The greater force of Ad-41 relative to fX174 at the same distance between virus and sand indicated that Ad-41 was more prone to deposition on goethitecoated sand, where the retention is irreversible. However, this is not in agreement with the results from the column experiments and AFM measurements. Column experiments show that fX174 was retained more than Ad-41 when they transported through the goethite-coated sand (Fig. 1b) and AFM force measurements indicate that fX174 experienced a greater adhesion force than Ad-41 (Fig. 2). We believe that the discrepancies between the measurements and the XDLVO estimations are because the XDLVO ignores morphological (e.g., roughness) effects and associated physicochemical heterogeneity of viruses and sand particles. The two viruses are not ideal spherical particles with smooth surfaces, and are composed of various protein macromolecules with different functional groups. Likewise, sand particles also are non-ideal in shape and have rough surfaces and charge heterogeneities, the roughness of sand surfaces may facilitate colloid deposition in shallow

S. Xu et al. / Water Research 123 (2017) 40e48

45

Fig. 3. Extended DLVO interactions between virus and sand.

primary minima that are susceptible to removal by diffusion (Shen et al., 2011). 3.2. Co-transport of the two viruses Breakthrough curves of the two viruses from their co-transport experiments through the goethite-coated sand are plotted in Fig. 4. Existence of Ad-41 in the influent promoted the transport of fX174 through the goethite-coated sand. fX174 showed little breakthrough (0.6%) in the absence of Ad-41, but its breakthrough percentage increased to 56.3%, 64.8%, and 70.6% when the concentrations of Ad-41 increased to 8.6  102, 1.1  104, and 4.8  104 TCID50/mL, respectively (Table 2). When the input concentration of Ad-41 increased from 1.1  104 to 4.8  104 TCID50/mL, the breakthrough percentages of both viruses increased (Table 2), indicating that the sand had limited sites to retain the viruses. A longer experiment of fX174 transport (70 PVs) was conducted to test this assumption. Fig. 5 shows that complete occupancy of the available attachment sites on the goethite-coated sand required at least 10 PVs of fX174 suspension (equivalent to 3.74  108 PFU of fX174 particles). Once the attachment sites were filled, retention of fX174 decreased sharply during 13e32 PV, and the sand had no significant further retention despite continuing injection of fX174 suspension from 32 to 70 PVs. The calculated maximum fractional surface coverage (qmax ¼ 0.5  10
4) of fX174 in the long-term experiment verified that only a small percentage of goethitecoated sand contributed to viruses retention (Table 3), suggesting

Fig. 5. Breakthrough of fX174 from goethite-coated sand in a long-term experiment.

that the goethite-coated sand had limited capacity to retain viruses. It is interesting to note that the sum of the fractional surface coverage (q) of fX174 and Ad-41 calculated from the short-term experiments equals the surface coverage value calculated from the long-term experiment with fX174 at corresponding pore volumes (Fig. 6). In addition, Fig. 6a demonstrates that the fractional

Fig. 4. Mutual promotion of transport of fX174 and Ad-41 through goethite-coated sand. 1  106 PFU/mL of fX174 was used in all experiments.

46

S. Xu et al. / Water Research 123 (2017) 40e48

Table 3 Calculated blocking factor (B) andqmaxvalues of viruses in goethite-coated sand column. Injection

fX174

Ad-41

Input Con. (PFU/mL) Long term Short term

6

1.0  10 1.0  106

qmax

B (
)

Naverage 2

(1/B,%) 6

2.0  10 3.6  106

(PFU/m )
4

0.5  10 0.3  10
4

11

1.2  10 7.2  1010

Input Con.

B

qmax

Naverage

(TCID50/mL)

(
)

(1/B,%)

(TCID50/m2)

e 4.8  104

e 5.0  106

e 0.2  10
4

e 4.5  109

Fig. 6. Fractional surface coverage of viruses on goethite-coated sand as a function of pore volume: (a) fX174 (1  106 PFU/mL) and Ad-41(4.8  104 TCID50/mL) co-existing in the influent in a short-term experiment and (b) fX174(1  106 PFU/mL) alone in the influent in a long-term experiment.

surface coverage of Ad-41 was higher relative to fX174. According to the calculated maximum fractional surface coverage (qmax ¼ 0.5  10
4) of fX174 in the long-term experiment (~6.55  1013 PFU of fX174) and assuming the same maximum area of sand surface could be occupied by both viruses, the maximum Naverage of Аd-41 was estimated to be 1.6  1010 TCID50/m2 (~8.73  1011 TCID50 of Аd
41), and thus additional input would lead to high virus breakthrough concentrations. This blocking effect on sand surfaces explains how Ad-41 promoted the transport of fX174, i.e., Ad-41 competed with fX174 for the limited electropositive attachment sites (<0.0005%) on goethite-coated sand surfaces to suppress the retention of fX174. In addition, once Ad-41 particles attached onto the sand surface, they became mostly irreversible and its larger surface area (relative to fX174) and the extending fibers could block adjacent accessible attachment sites via steric hindrance effect, reducing the retention of fX174. This is supported by the higher blocking factor B found for Ad-41 (5.0  106) compared to fX174 (3.6  106) in the co-transport experiment (Table 3). Similarly, the presence of fX174 promoted the transport of Ad41. The maximum effluent concentration C/C0 of Ad-41 (1.1  104 and 4.8  104 TCID50/mL) increased from 0.3 in the absence of fX174 to 0.50e0.55 at 106 PFU/mL of fX174 in the influent suspension (Fig. 4b) and accordingly increased the effluent percentages from 20% to 30%e34% (Table 2). This result indicates that the facilitated effect of fX174 on Ad-41 transport is less than that of Ad41 on fX174 transport. Two mechanisms might be responsible for the observed interactions of the two viruses. One is that there are limited sites on goethite-coated sand for virus attachment, and that Ad-41 is more efficient in competing for those sites than fX174 because an Ad-41 viral particle with its spiking fibers on its surface is ~4 times larger than a fX174 viral particle. The second possible mechanism is that fX174 may modify the surface charges of Ad-41 and sand grains. As mentioned earlier, the fibers of Ad-41 have high isoelectric points and are positively charged at the experimental pH in this study. The small, negatively charged fX174 might thus be

able to attach onto the large, positively charged sites on Ad-41, increasing the electro-negativity of Ad-41. As a result, fX174binding Ad-41 competed to attach on goethite-coated sand with coming Ad-41, contributing to increased Ad-41 transport. In addition, fX174 attachment on goethite could also decrease the surface potential of sand surfaces, reducing the attractive interactions between Ad-41 and the goethite-coated sand. 4. Conclusions The single and co-transport of human Adenovirus 41 and model bacteriophage fX174 were quantified with environmentally relevant porous media under saturated flow conditions. Surface coverage, adhesion force, and extended DLVO interactions between virus and porous media were estimated to analyze the interactive transport behaviors of the viruses. The main findings are: 1. Ad-41 behaves similarly in terms of transport and retention with model bacteriophage fX174 in non-reactive porous media, but very differently in reactive porous media. 2. Goethite-coated sand has a great potential to remove Ad-41 and fX174 from water, with a higher permanent removal suffered by Ad-41. 3. Ad-41 and fX174 could help each other to transport through reactive porous media, with the promotion effect of Ad-41 on fX174 transport more noticeable. 4. The main mechanism responsible for the mutual promotion of virus transport is competition for limited attachment sites and steric hindrance in relation to the existence of fibers on Ad-41. 5. Extended DLVO coupled with steric interaction cannot accurately predict virus transport behavior, owing to the theory's inability to take into account of morphology and heterogeneous surface properties of viruses. These findings have implications in the use of model bacteriophages (e.g., fX174) as surrogates for human pathogens (e.g, Ad-

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